The emergence and spread of resistance to current antimalarial therapies threatens to reverse gains made in reducing malaria related morbidity and mortality. Novel chemotherapeutic agents, along with increased insight into mechanisms of drug action and potential modes of resistance, are essential to counteract this threat. From over 500,000 small molecules screened against the asexual blood stage of Plasmodium falciparum in dose-response, we have assembled the NCATS Malaria Active Collection (NMAC). This collection of 1,339 compounds with potent activity against the asexual blood stage were used to profile the drug susceptibility response across the parasite life cycle, presenting a chemical biology snapshot of targetable pathways. In addition, using three Plasmodium genetic crosses, a chemical genomic approach was leveraged to gain insight into genetic loci modulating drug susceptibility. 204 compounds (15.2%) demonstrated significantly variant activity between the three parental lines (Dd2HB3, 91 compounds; 7G8GB4, 100 compounds; 803GB4, 42 compounds). Only four compounds were differentially active against all three comparisons. These include the antimalarials chloroquine and pyrimethamine, suggesting greater genetic complexity underlying drug response than previously observed.